The three mouse hedgehog (Hh) secreted proteins, Shh, Dhh, Ihh, are involved in many inductive processes during mammalian development, including ventralization of the DNS, limb patterning and testes development. Many of the genes in the signaling cascade downstream of hh in Drosophila have been identified and all have homologues in mammals. At the end of the pathway is a putative transcription factor, cubitus interruptus (ci). Hh induces ci protein and antagonizes the negative regulation of ci by the proteins patched and protein kinase A. In mammals, the homologues of ci are the three Gli genes, Gli1, Gli2, and Gli3. The overall goal of this research proposal is to determine whether the three mouse Gli genes have similar protein functions and whether they function in an Hh signaling pathway analogous to the Drosophila hh cascade. The specific aims are: 1. To determine whether expression of neural and limb developmental marker genes are differentially altered in Gli, Gli2 and Gli3 mutants. 2. To determine whether there is overlap in function between the Gli genes by studying the phenotypes of Gli compound mutants. 3. To determine whether Gli protein can substitute for Gli3 coding sequences with those of Gli using gene targeting. 4. To compare the effects of ectopic expression of Shh, human GLI and mouse Gli and Gli3 in transgenics. 5. To determine whether Gli genes regulate Shh expression by characterizing Shh DNA regulatory elements. The studies of the developmental defects of mouse Gli mutants and characterization of Hh genetic pathways will be directly relevant to human development and disease, as the genes are conserved in humans. Indeed, humans with the dominant Greig cephalopolysyndactyly syndrome have limb and craniofacial defects and spina bifida due to mutations in GLI3, and mouse Gli3 mutants display similar abnormalities. As well, humans with the dominant Gorlin's syndrome have a variety of tumors and diverse developmental defects and have mutations in PTC. Finally, the GLI gene is amplified in many human tumors.